Experimental infection of the cockroach Periplaneta americana with Toxocara canis and the establishment of patent infections in pups

The possible role of the cockroach Periplaneta americana in the transmission of Toxocara canis eggs and larvae via faeces and tissue migration was studied. Cockroaches fed with 3 x 105 and 5 x 105 embryonated eggs were found to harbour viable eggs and larvae from days 1 to 5 post-infection (DPI). At necropsy on 5 DPI, eggs and larvae were also recovered from the rectal contents but not from the tissues of cockroaches. In addition patent infections were established in pups fed on infected faeces of cockroaches, with eggs first appearing in the faeces of pups at 38 DPI. Adult worms of T. canis were also recovered at necropsy. Therefore the importance of cockroaches as good mechanical disseminators of ascarid eggs, especially T. canis, is discussed.     

Experimental infection of mice with Toxocara canis larvae obtained from Japanese quails

The migration and distribution of Toxocara canis larvae in the tissues of Japanese quails, infected orally with 5 x 10(3) infective eggs, were studied, as well as the re-infectivity of these larvae in mice, inoculated with 50 larvae obtained from the liver of these quails. Post-infection, the highest concentrations of larvae were found to be present in the liver of quails while only a few migrated to other tissues like lungs, heart, muscle and brain. The migration and distribution of the larvae in the tissues of mice were studied by necropsy on days 6 and 12 post-infection. On both days the highest number of larvae, 11 and 10, were recovered from the carcase followed by six and seven from the leg muscles and four and eight from the brain, respectively. A few larvae were recovered from the liver, lungs and viscera. This implies that the larvae had a special affinity for the muscle and brain tissue of mice, unlike in the quails. The role of these larvae in relation to paratenism is discussed.     

Pathological and haematological responses of cats experimentally infected with Toxocara canis larvae

Responses of eight adult cats to one or two infections with larvae of Toxocara canis were studied up to 39 days post infection (DPI). Clinically, all cats remained normal throughout the study. The major necropsy finding was multifocal, white to grey nodules mainly within the liver, lungs and kidneys; live larvae were found in liver nodules. Histologically, the nodules were eosinophilic granulomas. Granulomas containing a larval section were observed mainly within the liver. All infected cats had variably severe, eosinophilic arteritis and bronchiolitis and medial hypertrophy and hyperplasia of the pulmonary arteries. No inflammatory eye lesions were detected. Circulating eosinophil levels increased in all infected cats; peak values of 15,790 and 10,050 eosinophils microliters-1 were observed at 25 or 32 DPI in cats receiving a single or double infection, respectively. Bone marrow of all infected cats exhibited marked eosinophilic hyperplasia which did not correlate with the level of circulating eosinophilia. Thus, infection of cats by the larvae of T. canis causes disseminated eosinophilic and granulomatous disease with marked pulmonary artery and airway lesions.     

Larval recovery of Toxocara canis in organs and tissues of experimentally infected Rattus norvegicus

The aim of this note was to record for the first time the recovery of Toxocara canis larvae from tissues and organs of Rattus norvegicus (Berkenhout, 1769), Wistar strain, until the 60th day after experimental infection. Rats were orally infected with embryonated T. canis eggs, killed on days 3, 5, 8, 10, 15, 30, and 60 after inoculation and larvae were recovered from liver, lungs, kidneys, brain, and carcass after acid digestion, showing a pattern of migration similar of that previously observed in mice.     

Eosinophilia, granuloma formation, migratory behaviour of second stage larvae in murine Toxocara canis infection. Effect of the inoculum size

Experimental infection of mice with Toxocara canis provides one of the best models for immunological and pathological studies of the visceral larva migrans syndrome. Blood eosinophilia, the migratory behaviour of second stage larvae and granuloma formation were studied in Swiss mice infected with Toxocara canis. Eosinophilia, spleen, liver and lung indexes were followed during a primary infection with different inoculum sizes (500 and 1500 eggs) while the migratory behaviour of larvae was studied in a primary infection with 1500 eggs over a period of 4 months. In mice infected with three challenges of 1500 eggs in order to elicit a strong inflammatory reaction in the tissues, a histopathological study was carried out. The results showed that eosinophilia, spleen and lung indexes (but not the liver index) were influenced by the parasite inoculum size. The migratory behaviour study showed that larval recovery was maximal three days post-infection, from the liver and lungs; the peak recovery from the skeletal muscles and brain being on days 15 and 30 post-infection, respectively. The histopathological study revealed the formation of granulomas in all the tissues examined (liver, lungs, kidneys, spleen, lymph nodes, myocardium etc.) but not in nervous tissue or in the retina of the eye. Granulomas in the lungs were larger than those found in the liver. The implications of these results are discussed considering host-parasite inter-relations.     

Visceral larva migrans: migratory pattern of Toxocara canis in pigs.

The migratory pattern of Toxocara canis was investigated following infection of pigs with 60000 infective eggs. Groups of six pigs were slaughtered at 7, 14 and 28 days after infection (p.i.), and the number of larvae in selected organs and muscles was determined by digestion. A group of uninfected pigs was used as negative controls for blood parameters and weight gain. Toxocara canis migrated well in the pig, although the relative numbers of larvae recovered decreased significantly during the experiment. On day 7 p.i., high numbers of larvae were recovered from the lymph nodes around the small intestine and to some extent also from the lymph nodes around the large intestine, and from the lungs and the liver. On day 14, the majority of larvae were recovered from the lungs and the lymph nodes around the small intestine, and by day 28 p.i. most larvae were found in the lungs. Larvae were recovered from the brain on days 14 and 21, with a maximum on day 14 p.i. No larvae were found in the eyes. Severe pathological changes were observed in the liver and lungs, especially on day 14 p.i.; also, development of granulomas was observed in the kidneys. Finally, a strong specific antibody response towards T. canis L2/L3 ES products was observed from day 14 p.i. until termination of the experiment, and the maximum eosinophil response was observed 14 days p.i. The pig is a useful non-primate model for human visceral larva migrans, since T. canis migrate well and induce a strong immunological response in the pig. However, the importance of the pig as a paratenic host is probably minor, because of the relatively early death of most of the larvae.     

Experimental infection of pups with Ancylostoma caninum larvae from an abnormal host, the chicken

The migration and distribution of Ancylostoma caninum larvae in the tissues of chickens, infected orally with 1,000 larvae, were studied. Larval yield at necropsy from different organs after digestion with artificial gastric juice revealed a 62.9% recovery four hours after inoculation, followed by a sharp decline to 5.4% at 72 hours. Larvae were found in the heart within four hours, the lungs within eight hours and the liver within 12 to 18 hours but no larvae were recovered from the spleen, kidney or brain. Migration in the muscles of head, neck, thorax and abdomen was detected at 12 hours and was maximal at 36 hours. The establishment of patent infection in the definitive host was studied by feeding infected chicks to hookworm-free pups (one chick/pup) 48 hours, 7 days and 14 days after infection. The mean worm burden at necropsy was highest (15) in the pups fed with chicks 48 hours after infection and was three and nil in the other groups respectively.     

Migration behaviour and pathogenesis of five ascarid nematode species in the Mongolian gerbil Meriones unguiculatus

To understand the characteristic features of the Mongolian gerbil, Meriones unguiculatus, as an animal model of ascarid infections, the migration behaviour and pathogenesis of larvae were investigated in experimentally infected gerbils. Embryonated eggs from each of Toxocara canis, Baylisascaris procyonis, B. transfuga, Ascaris suum, and A. lumbricoides were orally inoculated into gerbils and larvae were recovered from various organs at designated periods. In T. canis-infected gerbils, larvae were present in the liver 3 days after infection and in the skeletal muscle and brain via the heart and lungs at a similar rate. In B. procyonis- and B. transfuga-infected gerbils, larvae were present in the lungs within 24 h after infection, with some having reached the brain by that time. After 24 h, larvae of B. procyonis tended to accumulate in the brain, while those of B. transfuga accumulated in skeletal muscles. In A. suum- and A. lumbricoides-infected gerbils, larvae remained in the liver on day 5 post-infection and elicited pulmonary haemorrhagic lesions, which disappeared 7 days after initial infection. Thereafter, no larvae of any type were recovered. Ocular manifestations were frequently observed in T. canis- and B. procyonis infected gerbils, but were rare in B. transfuga-infected gerbils. In the cases of A. suum and A. lumbricoides, migration to the central nervous system and eyes was extremely rare, and larvae had disappeared by 2 weeks post-infection. Fatal neurological disturbances were observed in B. procyonis-infected gerbils, whereas irreversible non-fatal neurological symptoms were observed in the case of B. transfuga.     

Longevity of Toxocara cati Larvae and Pathology in Tissues of Experimentally Infected Chickens

This study was conducted to determine the distribution patterns and duration of stay of Toxocara cati larvae in organs of chickens and to investigate chronic phase and potential zoonotic risk of toxocariasis in chickens. Chickens were orally infected with 1,000 embryonated T. cati eggs and necropsied 240 days post-infection. Organs of the chickens were examined at gross and microscopic levels; tissues were digested to recover larvae. Peribronchiolitis with infiltration of lymphocytes, and hyperplasia of bronchiolar associated lymphatic tissues (BALT) and goblet cells, were evident in the lungs of infected chickens. There were mild hemorrhages and infiltration of lymphocytes and a few eosinophils in the meninges. Larvae were recovered from 30% of the exposed chickens. Larvae recovery indicated that T. cati larvae stay alive for at least 240 days in the chicken brain. Therefore, chickens may potentially act as a paratenic host in nature and transfer T. cati larvae to other hosts.     

The use of 35S-labelled Toxocara canis to determine larval numbers in tissues.

The potential of using 35S-labelled larvae to determine the number of second-stage Toxocara canis larvae present in the tissues of infected animals was assessed. Infective larvae were labelled by in vitro culture with medium containing 35S-methionine. The amount of radiolabel attached to larvae decayed exponentially with time and had an in vitro mean half life of 3.54 +/- 0.65 days. The 'lost' radiolabel was incorporated into proteins which formed part of the worm's excretory/secretory products. The levels of radioactivity present in different organs of BALB/c mice, infected with 35S-labelled T. canis larvae, varied over the course of infection. Initially most of the radioactivity was present in liver, but over the course of infection 35S liver levels gradually decreased and brain levels increased. By day 14 post-infection the majority of the isotope was present in the brain (p < 0.01). Assessment of antibody levels on day 14 post-infection showed that infection with 35S labelled T. canis larvae induced the production of parasite-specific IgM, IgG and IgG1 antibodies.     

Eosinophil peroxidase levels in hearts and lungs of mice infected with Toxocara canis

Toxocara canis infection of abnormal hosts results in a condition in which infective larvae migrate through the soft tissues of the body, exclusive of the skin. This condition is known as visceral larva migrans (VLM) and causes a syndrome characterized by hepatosplenomegaly, hyperglobulinemia, hypereosinophilia, and transient pulmonary infiltrates. Because of the known association between hypereosinophilia and eosinophilic heart disease, we have been studying the hearts of mice infected with T. canis for evidence of myocardial damage and have previously described a severe eosinophilic myocarditis that leads to a marked myocardial fibrosis. We have measured eosinophil peroxidase (EPO) levels (a marker enzyme for specific granules of eosinophils) in homogenized lungs, homogenized hearts, and eosinophils recovered from the lungs of mice infected with T. canis over a 6-wk period. A marked accumulation of EPO was observed in the lungs of infected mice from day 14 postinfection (PI) to at least 6 wk of infection. Most of the EPO was associated with eosinophils that comprise the bulk of the pulmonary infiltrates associated with the VLM syndrome. However, following bronchoalveolar lavage, cytochemical localization of EPO activity in lungs from infected mice suggested that eosinophil degranulation had resulted in this marker enzyme being deposited within the pulmonary parenchyma. Peak levels of EPO were found in the myocardium by day 14 PI and declined over the 6-wk period. These levels equaled about 1/3 of the levels seen in the lungs of the same mice. These studies suggest that in mice infected with T. canis, the presence of increased numbers of eosinophils may lead to marked peroxidatic cardiopulmonary damage.     

Brugia pahangi: effects of duration of infection and parasite burden on lymphatic lesion severity, granulomatous hypersensitivity, and immune responses in jirds (Meriones unguiculatus)

The effects of Brugia pahangi infection duration and parasite burden on parasite-associated inflammatory and immune responses were determined over a 181-day period in jirds receiving from one to eight inoculations of infective larvae. Multiple infections did not produce a protective resistance to reinfection as determined by adult worm recovery at necropsy. Intralymphatic granulomatous lesions, lymph thrombi, were first seen at 48 days post initial inoculation (DPI). The numbers of lymph thrombi reached peak levels in singly inoculated jirds at 90 DPI and significantly decreased to low levels by 160 DPI. The ratio of lymph thrombi to adult worms recovered from the spermatic cord lymphatics followed a similar pattern. Sizes of renal lymph nodes, which drain lymphatics containing parasites, followed a temporal pattern of increase and decrease similar to that of lymph thrombi numbers. Peak granuloma areas around antigen-coated beads embolized in lungs were seen at 27 DPI. Granuloma areas around antigen-coated beads began to decrease after 69 DPI and reached sizes not significantly different from uninfected controls by 118 DPI. Multiple inoculations of infective larvae and increasing worm burdens did not affect the pattern of granulomatous response to antigen-coated beads. Eosinophilia of singly and multiply infected jirds peaked at 26 DPI. Eosinophilia of singly infected jirds returned to normal levels by 103 DPI but those of multiply infected jirds remained elevated until 160 DPI. Lymph node cell blastogenic responses to antigen were greater than those of splenocytes at all time intervals measured. However, significant differences in stimulation indexes between groups with different infection durations were not seen with either cell type. Antibody responses to somatic adult worm antigen as measured by ELISA reached near peak levels by 48 DPI and remained elevated for the course of the study in all infected jirds. The decrease in lymphatic lesion severity seen in chronically infected jirds temporally corresponds to the decrease in granulomatous reactivity measured around antigen-coated beads embolized in the lungs. This observation suggests that host and/or parasite factors associated with these two phenomena may be similar. Although these decreases may be the result of down-regulated immune responses, corresponding decreases in antibody levels and blastogenesis of lymphocytes stimulated by crude worm extracts were not observed in chronic infections.     

Effects of irradiation on the biology of the infective larvae of Toxocara canis in the mouse

Mice were infected with either 2,000 normal or irradiated embryonated eggs of Toxocara canis and the number of larvae in their livers, lungs, brains, and carcasses investigated at 5, 20, and 33 days of infection. Mortality of mice infected with normal eggs was 33% between day 4 and 8 postinfection but there was no mortality among mice infected with irradiated eggs. Irradiation with 60, 90, or 150 kr of X-rays inhibited the migration of larvae from the livers and lungs and their accumulation in brain and carcass in proportion to the irradiation dose. By day 33 of infection, the ratio of larvae in liver and lungs to larvae in brain and carcass was 0.16 in normal mice, 0.42 in 60-kr mice, 0.98 in 90-kr mice, and 23.3 in 150-kr mice. Irradiated larvae, particularly those migrating through the peritoneal cavity, died faster than normal larvae until day 20. Irradiation favored survival after day 20. By days 20 and 33 postinfection the total parasite load was 29% and 8%, respectively, of the administered dose in control mice, 18% and 12% in 60-kr mice, 8% and 4% in 90-kr mice, and 0.9% and 0.3% in 150-kr mice. Irradiation of infective T. canis larvae, then, reduces their pathogenicity, inhibits their migration from liver and lungs, kills some of the parasites during the first 3 weeks of infection, but favors their late survival in the host.     

Aberrant Toxocara canis in a red fox

During necropsy of a red fox (Vulpes vulpes) heart an adult, male Toxocara canis was found under the pericardium at the junction of the right ventricle and right atrium. The life cycle of T. canis is complex and includes tracheal and somatic migrations of larvae, and they can be found in many tissues throughout the host's body. However, it is rare for adult ascarids to be recovered outside of the small intestine. This is the first report of an adult T. canis inside the pericardial space.     

Inhibition of inducible nitric oxide synthase in murine visceral larva migrans: effects on lung and liver damage

The roles of nitric oxide production and oxidative process were studied in mice infected with Toxocara canis and treated with aminoguanidine which is a specific inhibitor of inducible nitric oxide synthase (iNOS). Relations of nitric oxide synthase inhibition and tissue pathology were assessed by biochemical, histological and immunohistochemical methods. In experiments, Balb/c albino mice were inoculated with T. canis eggs either with or without aminoguanidine treatment or alone, at 24th, 48th hours and on 7th days. LPx and SOD values in liver tissue and plasma were measured. Liver and lung tissues were evaluated for the pathological lesions. The expression of eNOS and iNOS in both tissues were studied with immunohistochemistry in the same intervals. We observed significant differences between T. canis infected and aminoguanidine treated animals. Larval toxocarosis led to oxidative stress elevation in plasma. Microscopic examination of the liver histological sections revealed pathological lesions in the hepatic parenchyma in infected mice. In the mice received T. canis eggs plus aminoguanidine, the sinusoidal areas were enlarged. Histological lesions were more severe at 48 hours after infection. Numbers of eNOS and iNOS expressing epithelial cells were increased in the T. canis infected mice. The activities of eNOS and iNOS were also observed in the body of the larvae which have migrated to lung and liver. As a result, we have demonstrated that in vivo production of eNO and iNO during T. canis infection cause direct host damages and it is strongly related to the oxidative stress. We propose that larval NO can also be effective in larval migration, but it needs further investigation on distribution of NO in larvae.     

Initial stage of development and migratory behavior of Toxocara canis larvae in BALB/c mouse experimental model

In the present study, the initial developmental stage of Toxocara canis eggs and larvae, and number of recovered larvae from BALB/c mouse-infected organs are described. In vitro culture of T. canis detects the frequencies of interphasic, mitotic and embryonated eggs only within a 7-day period. Analysis by egg counting was carried out for 32 days. The results showed that at 7 days after cultivation, the frequency of larvae was 50.4% and that this frequency reached 52.8% in 32 days. In the experimental infection of BALB/c mice with T. canis, the number of recovered larvae statistically increased in the brain and liver, with doses of approximately 200 and 1000 eggs. After 7 days of infection, a larger number of larvae were obtained in the lung and liver, although a maximum amount was found in the brain after a 15- or 30-day post-infection period.     

Regulation of toxocariasis in mice selectively reared for high and low immune responses to Nematospiroides dubius

Test mice have been selectively reared for high (H) or low (L) immune responses to Nematospiroides dubius. After secondary infection with N. dubius, the L mice voided ten times as many eggs in their faeces as the H mice, and at necropsy, 71% versus 20% of the inoculum of N. dubius were recovered as adult worms from the L and H mice respectively. Furthermore, N. dubius were more fecund in the L than in H mice. High or low immune responsiveness was not restricted to N. dubius infection in these mice but was also observed during Toxocara canis infection. The migration of T. canis larvae from gut via the liver to skeletal muscle and CNS was inhibited in H versus L mice. Many more larvae were recovered from the livers of H compared with L mice which was indicative of greater immunity in the H mice. The protective immune response in H compared with L mice to both N. dubius and T. canis included pronounced eosinophilia and elevated antiparasite antibody titres.     

Experimental life cycle of Lagochilascaris major leiper, 1910 (Nematoda: Ascarididae) in cats (Felis domesticus)

The life cycle of Lagochilascaris major was studied using eggs collected from a natural clinical case in a domestic cat. Twenty-seven white mice (Mus musculaus), 5 hamsters (Mesocricetus auratus), and 1 vesper mouse (Calomys callosus) were orally inoculated with 800-1,300 embryonated eggs. When examined from 73 to 246 days postinoculation (PI), encysted third-stage larvae were seen in skeletal muscles and less frequently in connective tissue, liver, and lungs. Twenty-two of the 23 cats orally inoculated with 40-430 encysted larvae from these rodents, and necropsied from 1 hr to 185 days PI, became infected. Third-stage larvae were located in the stomach, esophagus, and oropharynx from 1 to 24 hr PI. At 48 hr, larvae, from mainly the fourth stage, were only found, unilaterally or bilaterally, inside a "sac" in the region of the semilunar fold of the palatine tonsil at the base of the tongue. Adult worms were found in this location from 10 to 175 days PI. No fistulated abscess to the outside medium was found. Adult worms were also found in the middle ears of 2 cats showing purulent otitis. Eggs in the ear secretion were under different stages of development. Eggs in feces were first observed on days 14 and 15 PI, and 1 cat shed them until 178 days PI. Six infected cats were treated with fenbendazole at 50 mg/kg of body weight for 3 consecutive days, eliminating all the parasites present in the tonsils. The drug was not effective against the parasites present in the middle ear. No stage of the parasite was found in the tissues of 5 cats given 4,000-5,200 eggs orally and examined after 19 and 50 days PI. This indicates that the life cycle of L. major requires an obligate paratenic host and is characterized by heteroxenic cycle.     

Toxocara canis: search for a potential drug amongst beta-carboline alkaloids--in vitro and mouse studies

The goal of this study was to search for new treatments for Toxocara canis using both in vitro and in vivo experiments. We specifically looked for a treatment for T. canis larva migrans, and examined beta-carboline alkaloids (17 compounds) with various structural modifications, both in in vitro and in vivo experiments. In the in vitro experiments, screening for nematocidal activity on the T. canis second stage larvae, cytotoxic activity, and immune activity in the host were undertaken. Compound 17 was selected, as it exhibited nematocidal activity for T. canis larvae and did not have any cytotoxic or immunosuppressive activity in the host. The effectiveness of compound 17 was then examined using T. canis larvae infected mice in in vivo experiments. To evaluate the anthelmintic effect, the relative mobility value for the larvae was examined in addition to the number of larvae in the brain, skeletal muscle, and liver. Compound 17 was also examined in both free and liposome-entrapped (LE) forms. Polyethylene glycol (PEG)-LE compound 17 showed an anthelmintic effect in which the number of larvae in the brain was decreased compared free albendazole. PEG-LE compound 17 also effectively suppressed the mobility of the larva in brain and skeletal muscle. The experimental procedure employed assisted in the discovery of this potential candidate and is a promising approach for finding alternative therapeutic regimens for T. canis larva migrans.     

Brugia pahangi: in vivo tissue migration of early L3 alters gene expression

Events occurring during early filarial nematode migrations are central to parasite establishment but rarely studied. Brugia pahangi larvae injected intradermal (ID) into the hind limb of the gerbil (Meriones unguiculatus) can be recovered from the popliteal lymph node (POP) at 3 days post-infection (DPI). They have been designated migrating larvae (IDL3). Alternatively, L3 recovered at 3DPI from the peritoneal cavity (IPL3) do not migrate. Subtracted cDNA libraries using IDL3 and IPL3 revealed distinct gene profiles between IDL3 and IPL3. Troponin-c was significantly upregulated in IDL3, while Cathepsin L was significantly increased in IPL3. Differences in mRNA levels were also observed with these and other genes between IDL3, IPL3 and L3 isolated from mosquitoes (VL3). These data suggest that migratory activity, exposure to potentially different host environments and/or host location may be important external factors in influencing larval gene expression.